Metabolite screen Microbacteria
Thoenen et al. AMPO formation
Lisa Thönen
2024-05-21
Here we plot the results of the metabolisation assay in liquid cultures of Microbacteria from the maize root bacteria (Thoenen et al. 2023) and the AtSphere collection (Bai et al. 2015). We tested MBOA and DIMBOA-Glc. Additionally we also report growth data of all Microbacteria in MBOA-containing half-strength TSB medium to test them for MBOA tolerance and in MBOA-containing minimal medium to test them for using MBOA as sole carbon source for growth.
MBOA metabolisation
Quantification & calculations: using a standard curve the samples are quantified
Prepare standard curves
Calculate the concentrations
Quick overview of measured/detected BXs
Phylogenetic tree
Prepare tree with information on host origin
Add AMPO phenotype on plates
Add quantitative MBOA & AMPO phenotypes
Prepare orthogroup data for the 5 OGs that show 100% sensitivity and specificity for AMPO phenotype on plate
Plot AMPO phenotype in plate assay along with quantitative MBOA and
AMPO concentrations measured for liquid cultures grown in half-strength
TSB supplemented with 500 uM MBOA. Orthogroups showing 100% sensitivity
and specificity are shown on the side. Note that KHB019 was not assessed
in liquid culture and therefore is shaded in grey for MBOA and AMPO.
Figure S8a: BX compounds measured after incubation in half-strength
TSB supplemented with 500 uM MBOA.
DIMBOA-Glc metabolisation
Quantification & calculations: using a standard curve the samples are quantified
Prepare standard curves
join 11 stds and HMPAA
remove the compounds which are not reliable quantified
color code Compounds
Dilutions: 150 + 350 = 500 50 + 700 = 750 500 / 150 = 3.33333 750 / 50 = 15
1/((500/150)*15) = 0.02
for some reason the MBOA concentrations are a bit too high (factor 1.273141 controlled with t0 MBOA sample) –> ev because of precipitaion issue?
Explore raw data
Phylogenetic tree
Figure S8b: BX compounds measured after incubation in half-strength
TSB supplemented with DIMBOA-Glc
Metabolite categories for comparative genomics
MBOA
| max_MBOA | max_AMPO | max_HMPAA |
|---|---|---|
| 516.2433 | 302.6399 | 44.06674 |
DIMBOA-Glc metabolization
| max_DIMBOA_Glc | max_MBOA | max_AMPO |
|---|---|---|
| 575.2392 | 120.9501 | 7.355668 |
Groups
Growth Microbacteria TSB (Metabolite samples) & MM (Carbon source)
Growth curves of all plates single treatments
AUC
To quantify the total bacterial growth over time, we calculate the total area under the curve. This is done with the function “auc()” For comparison between treatments, the total AUC (calculated using density increase) and AUC_raw (calculated using raw density) is normalized with the AUC of the strain grown in the control treatment (no chemicals added, just normal growth media with DMSO).
Plotting growth curves of minimal media
3 replicates (Rep) of each strain per plate 2 replicates (Replicate) per plate (twice the identical plate per run) 2 identical runs
Phylogenetic tree
Figure S8c
Combined tree
Thresholds for qualitative analysis - weak MBOA-degraders: >30% of MBOA degraded compared to the control) - strong MBOA-degraders: (>90% of MBOA degraded, Fig. 3a). strong AMPO-formers (100 - 10 %) weak AMPO-formers’, <10% of max. AMPO forming strain) (0.01 µM, limit of detection).
Tree with AMPO Phenotype on plates
Tree as above with MBOA metabolites
Tree as above with DIMBOA-Glc metabolites
Tree as above with carbon source data
Tree as above with orthogroups
Figure 3 note: phenotype of KHB019 on plate was adjusted in Illustrator to no AMPO.
## R version 4.4.0 (2024-04-24)
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